Defects in mitochondrial gene expression can cause a myriad of mitochondrial disorders, including mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, Kearns-Sayre syndrome, and Leber hereditary optic neuropathy. These diseases have a large impact in the population since, when considered as a class, they are estimated to have a prevalence of at least 1 in 5000. Furthermore, a progressive decay in mitochondrial function is associated with human aging and mitochondrial deficiencies are thought to contribute to the onset of age-related diseases. Clear associations exist between deficiencies in mitochondrial function and neurodegenerative diseases such as Alzheimer's and Parkinson's disease, as well as diabetes and cancer. There is extensive evidence linking mitochondrial deficiencies to human pathology, and in particular of the central role that defects in mitochondrial gene expression play in pathogenesis. A crucial requirement for mitochondrial gene expression is proper ribosome biogenesis. This process, which is not well understood, involves several posttranscriptional RNA modifications that are thought to be crucial for ribosome assembly. KsgA and Dim1 methyltransferases dimethylate two adjacent adenine residues in a conserved stem-loop in the small subunit of the bacterial and eukaryotic ribosomes, respectively. Both the stem-loop structure and methylation events are conserved in the human 12S mitochondrial rRNA. Mitochondrial transcription factor B1 (TFB1M) is essential for mammalian development and catalyzes the analogous rRNA modification in mitochondria. Methylation by KsgA is related to bacterial aminoglycoside sensitivity and TFB1M activity could similarly be related to the effect of aminoglycoside antibiotics on the mitochondrial ribosome and aminoglycoside-induced hearing loss. Consistently, aminoglycoside sensitivity in humans is often maternally inherited, and methylation activity by TFB1M modulates the effects of a pathogenic mtDNA mutation linked to deafness. Other TFB1M polymorphisms are associated with reduced insulin secretion and increased risk of type II diabetes mellitus, further highlightin the importance of this modification for normal mitochondrial function. Metazoan mitochondria contain a second TFB family member, TFB2M, in addition to TFB1M. TFB2M was discovered along with TFB1M as a putative mitochondrial transcription factor that associates with the mitochondrial RNA polymerase and promotes initiation of mitochondrial transcription. It is now accepted that TFB2M is essential for the initiation of transcription in mitochondria, yet surprisingly, neither TFB1M nor TFB2M display similarity to any known transcription factor, raising the question of why a protein with a predicted methyltransferase fold plays a central role in transcription initiation. This question remains unanswered, since the mechanism by which TFB2M promotes initiation is not yet known. Both TFB1M and TFB2M are predicted to adopt methyltransferase folds, yet their atomic structures remain unknown. Key structural differences must exist between them that explain their evident functional divergence. The goal of this proposal is to provide a more complete understanding of how TFB proteins influence disease pathogenesis by elucidating the roles that they play in mitochondrial gene expression.